Characterization of ultrasonically extracted flaxseed polysaccharide gum and assessing its lipid‐lowering potential in a rat model

Abstract Flaxseed polysaccharide gum (FPG) was extracted through the ultrasound‐assisted process using water as a solvent with a yield ranging from 8.05 ± 0.32% to 12.23 ± 0.45% by changing different extraction variables. The extracted FPG was analyzed for its functional groups and antioxidant potential. The maximum DPPH (2,2‐diphenyl‐1‐picrylhydrazyl) free radical scavenging activity (≈100%) of FPG was noted at concentrations beyond ≈10 mg·ml−1. The maximum inhibition percentage through ABTS (2,2′‐azino‐bis 3‐ethylbenzothiazoline‐6‐sulfonic acid) (72.4% ± 1.9%) was noted at 40 mg·ml−1, which was observed to be less when compared to DPPH at the same concentration. The total antioxidant potential of the FPG solution at a concentration of 10 mg·ml−1 was equivalent to 461 mg ascorbic acid, which tends to increase with concentration at a much lower scope. The in vivo trial suggested that the least weight gain was noted in experimental groups G2 and Gh2. A significant reduction in total cholesterol was noticed in G1 (−14.14%) and G2 (−17.72%) and in Gh1 (−22.02%) and Gh2 (−34.68%) after 60 days of the trial compared to the baseline values. The maximum reduction in total triglyceride was observed in Gh2 (−25.06%) and Gh1 (−22.01%) after 60 days of the trial. It was an increasing trend in high‐density lipoprotein cholesterol (HDL‐c) in different experimental groups G2 (10.51%) than G1 (5.35%) and Gh2 (48.96%) and Gh1 (31.11%), respectively, after 60 days of study interval. Reduction of −5.05% and − 9.45% was observed in G1 and G2, while similar results were observed in Gh1 and Gh2. Conclusively, results suggested a possible protective role of FPG against hyperlipidemia.


| INTRODUC TI ON
Recent evidence has shown the beneficial role of plant polyphenols in preventing and treating various metabolic syndromes, including diabetes, cancer, obesity, stroke, hyperlipidemia, and cardiovascular complications (Liu, 2013). The lifestyle modifications and changes in dietary patterns may significantly reduce the disease burden in one's life. Plant-derived natural compounds include lignans, polysaccharides, stilbenes, flavonoids, xanthophyll, phenolic acids, carotenes, flavonols, tannins, etc., abundantly found in various parts of plant matrices (Manzoor, Ahmad, et al., 2019;Xu et al., 2017). Among primary and secondary constituents present in plants, polysaccharides are known as key biological macromolecule that consists of homoand hetero monosaccharides and uronic acid linked via glycosidic bonds. Bioactive polysaccharides are abundant in different parts of seaweed, plants, bacteria, fungi, and animals and are responsible for performing various physiological functionalities in a living organism (Ullah et al., 2019).
Extraction of bioactive components from plant-based materials is vital and can be achieved via different techniques. The most efficient approaches include solvent extraction, hydro-distillation, microwave-assisted extraction, supercritical fluid extraction, and extraction through ultrasonication. Each technique has its own advantages and disadvantages (Manzoor et al., 2020;Samaram et al., 2015).
Among these, ultrasonication has several advantages over other extraction techniques like low temperature and energy requirements, lower extraction time, and the quality of extracted material Ahmed et al., 2020). Ultrasonication disrupts plant tissues via physical forces developed during the cavitation process, which results in an effective release of extractable constituents in a short time Manzoor, Hussain, et al., 2021;Manzoor, Hussain, Naumovski, et al., 2022;Umego et al., 2021).
Phytochemicals like polyphenols, carotenoids, aromas, and polysaccharides have been successfully extracted through ultrasonication from plant materials. Studies have shown positive effects of ultrasonication for the extraction of specific compounds, such as antioxidant compounds, polysaccharide gum, etc., using novel techniques from plant food (Chemat et al., 2020;Kumar et al., 2021;Manzoor, Hussain, Tazeddinova, et al., 2022;. Flaxseed (Linum usitatissimum L.) is gaining more importance as a functional food throughout the world due to its high content of soluble and insoluble dietary fibers, linolenic acid, and lignans (secoisolariciresinol diglucoside [SDG]), which are involved in the management of cholesterol (Naik et al., 2018;Soltanian & Janghorbani, 2018).
Lignans derived from flaxseed play a pivotal role in managing atherosclerosis, including potential as potent angiogenic, antiinflammatory, antioxidant, and anti-apoptotic properties (Kezimana et al., 2018;Zanwar et al., 2012). Most importantly, α-linolenic acid (ALA) present in flaxseed oil is also associated with human health advantages (Baker et al., 2016). Fibers from flaxseed have a significant role in managing serum cholesterol and serum glucose levels by reducing or otherwise delaying the absorption from the intestine (Dzuvor et al., 2018;Repin et al., 2017).
The most common method for the extraction of polysaccharide gum from different sources is aqueous extraction which is not considered time-and energy efficient. So, the best alternative technique is ultrasonication due to its simplicity, cost efficiency, and optimum extraction yield with fixed biological properties. This study was designed to extract polysaccharide gum from flaxseed through ultrasonication. Furthermore, the extracted polysaccharide gum was evaluated for its lipid-lowering potential in an animal model.

| Procurement of raw materials
The flaxseed (Linum usitatissimum) cv. Chandni was acquired from Oilseeds Research Center, Ayub Agriculture Research Institute, Faisalabad, Pakistan. Proper cleaning of seeds was done to remove any extraneous matters. Oil extraction was carried out by Mini Oil Presser (Model 6YL-550) at low temperature with a 2-3 kg/h capacity to obtain a partially defatted flaxseed meal. The flaxseed meal was stored until further processing at 25 ± 2°C in double zipper plastic bags of 1-kg capacity.

| Ultrasound-assisted extraction of flaxseed polysaccharide gum (FPG)
Ultrasound-assisted extraction of FPG was performed using ultrasonic (model VCX 750, Sonics & Materials, Inc.) with distilled water as the solvent by optimizing different extraction conditions (Zhong & Wang, 2010). Filtration of extracted FPG solution was carried out through a 40-mesh screen. The filtered material was then precipitated with 95% ethanol. Centrifugation was done to separate FPG from solution, and the FPG yield was calculated as a percentage of partially defatted flaxseed meal. Alkaline titration was used to calculate cyanogenic compounds in the form of hydrocyanic acid (HCN) present in FPG samples according to the procedure described in AOACC (2019). To determine tannins in the FPG sample, Schanderi (1970) adopted the method.

| Fourier transform infrared spectroscopic analysis
FPG sample was analyzed for its functional group through Fourier transform infrared (FTIR) spectroscopy with the help of Analect Instrument fx-6160 (Irvine, CA, USA), as illustrated by Sila and coworkers (Sila et al., 2014). After mixing 1 mg of FPG sample with 100 mg potassium bromide (KBr), the transmission (%) was recorded between 1000 and 4000 cm −1 . The similarities of the FPG sample were compared with those of the FTIR spectrum of gum arabic as standard.

| DPPH free radical scavenging activity
The capacity of polysaccharide gum to scavenge DPPH free radicals was determined by the methods illustrated by Koubaa, Ktata, et al.,  (1-20 mg) of FPG with 0.5 ml distilled water and 0.5 ml absolute ethanol without DPPH solution. The following equation was used to estimate the free radical scavenging activity (% inhibition).

| ABTS free radical scavenging activity
ABTS (2,2′-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid) was used to determine the free radical scavenging activity of FPG solution based on the discoloration of cations (Re et al., 1999). A mixture was made with 7 μM ABTS using distilled water, and a solution of 2.45 μM potassium persulfate was added. The prepared solution was stored at normal temperature for a time duration of 16 h. The ABTS •+ radical cations resulted in an intense color. The mixture was diluted with distilled water, and the absorbance was measured at 734 nm. The reaction mixture was kept at room temperature for 6 min, and then 10 μl of FPG solution at different concentrations was mixed with 1 ml of ABTS •+ diluted solution (A 734 nm = 0.7 ± 0.02). It shows the antioxidant molecules' capacity to prevent ABTS radical oxidation. The following equation was calculated to ABTS •+ scavenging activity in terms of % inhibition of FPG solution.
where A: Solutions containing the free radical ABTS •+ and A 0 : Solutions without the free radical ABTS •+ .

| Total antioxidant activity
The total antioxidant activity of FPG was determined by the method used by Koubaa et al., 2015 with some modifications. Different concentrations of FPG (5, 7.5, 10, and 15 mg) were taken in tubes, and then 1 ml of reagent solution (4 mM ammonium molybdate, 28 mM sodium phosphate, and 0.6 M sulfuric acid) was added. Distilled water was used up to 1.1 ml to make the volume in each tube, and all tubes were kept in a thermostatic water bath by maintaining a higher temperature of 90°C for 1.5 h. Afterward, the tubes were cooled down to room temperature, the total antioxidant activity was measured at 695 nm, and a standard curve was used to express it as ascorbic acid equivalent (AAE). Similarly, using the above procedure, a blank reading was taken by adding 1 ml of the standard reagent with 100 μl distilled water.

| Experimental animals and diets
Ethical guidelines of the parent institute were followed for approval of the study design. For this research, male rats (age 7-8 weeks, body weight ranging from 160 ± 10 g) were acquired from the National Institute of Health, Islamabad, Pakistan. To normalize with conditions, the rats were kept in properly designed cages under controlled conditions of a temperature of 25 ± 2°C and relative humidity of 60 ± 5% with proper ventilation and a 12 h light-dark cycle.
Standardized feed was prepared to contain 65% starch, 10% corn oil, 10% casein, 3% salt mixture, and 1% vitamins' mixture, and all groups of rats were fed with a basal diet. On the other hand, adding 10% cellulose and 1% cholesterol for hyperlipidemia is used in the standard diet. For baseline serum profile, the rats (n = 10) were slaughtered at the start of the study. Different biomarkers of total cholesterol, triglycerides, low-density lipoprotein cholesterol (LDLc), and high-density lipoprotein cholesterol (HDL-c), confirmed hyperlipidemia in experimental rats. Then, normal experimental group G 0 was given a basal diet, whereas other normal rat groups G 1 and G 2 were given diets comprising of FPG at 125 mg and 250 mg, respectively, up to the study trial of 60 days.
Similarly, hyperlipidemia-induced rat group G h0 was given a basal diet, whereas other hyperlipidemic rat groups, G h1 and G h2 , were given diets comprising FPG at 125 and 250 mg, respectively, up to the study trial of 60 days. Each of the groups mentioned above consisted of 10 experimental rats. Feed utilization by each experimental rat was estimated by subtracting the remaining spilled diet from the total quantity of diet supplied each day (Wolf, 2003). Body weight gain in experimental rats was observed after 30-and 60-days' duration.

| Serum collection
Blood samples were collected on days 0, 30, and 60 through the cardiac puncture technique, and the method of Uchida et al. (2001) was used for collecting blood serum samples.

| Serum lipid profile
CHOD-PAP method was used to determine serum cholesterol levels, as illustrated by Stockbridge et al. (1989). Annoni et al. (1982) used a method to estimate the levels of total triglycerides in all serum samples by liquid triglycerides (GPO-PAP). McNamara et al. (1990) employed the method to determine the levels of low-density lipoprotein cholesterol (LDL-c). High-density lipoprotein cholesterol (HDL-c) precipitant method was employed to estimate the HDL-c level in serum samples, as previously described by Assmann (1979).

| Statistical analysis
Triplicate readings were carried out for all the analyses, and average values were measured to interpret the results. Duncan's multiple range (DMR) test was used to estimate the significance level among the calculated mean values. Triplicate readings were recorded for biochemical parameters, and significant differences were measured at 5% significance using statistical software Minitab version 14.1 (Steel & Torrie, 1997).

| FTIR characterization
The ultrasound-assisted extracted FPG was characterized for its
FPG exhibited good scavenging activity compared with BHA, showing its capacity to react with free radicals and then convert to simpler and more stable products.

| ABTS free radical scavenging activity
ABTS has similar scavenging potential to that of DPPH free radical.
More stable compounds from free radicals have also been formed through ABTS, just like DPPH. Results for ABTS free radical scavenging activity are represented in Figure 2b, which shows that the concentration of FPG in solution has a proportional effect on the free radical scavenging activity. The maximum inhibition percentage (72.4% ± 1.9%) was noted at 40 mg·ml −1 , which was less when com-

| Total antioxidant capacity
Total antioxidant capacity (TAC) was estimated by reducing the phosphomolybdate. At acidic pH, the green complex of phosphate/ Mo (V) formed, and its absorbance was noted at 695 nm spectra. As

| Effect on weight
In this research, a significant reduction in body weight was observed in the hyperlipidemic group of experimental rats compared to the normal group of experimental rats (Figure 3). Maximum weight gain in normal experimental rats was noted in G 0 followed by G 1 , whereas on the other hand, in G 2, the lowest increase was observed after 30 days of studies. For normal experimental rats, similar observations were recorded after 60 day of trials. Almost similar observations were recorded for hyperlipidemic groups of experimental rats as the maximum weight gain was observed in Gh 0 , while the experimental group Gh 2 showed the lowest weight gain after 30 days of study. Similarly, after 60 days of trial, the maximum gain in weight was recorded in Gh 0 followed by Gh 1, and the least gain in weight was noted in Gh 2 .
The current research showed that FPG could reduce body weight in experimental rats due to its special dietary fiber ingredients. The FPG possibly improves the absorption of water, thus resulting in increased intestinal volume, constipation, and ease in defecation, ultimately resulting in weight management (Singh et al., 2011).

| Effect on total cholesterol
The experimental groups of normal and hyperlipidemic rats were observed for their serum lipid profile during the research trial after 30-and 60-day time intervals ( Table 2). The maximum significant reduction of −9.39% was noted in group G 2 in normal experimental rats after 30 days of the study trial, while a significant reduction of −8.23% was observed in G 1 of experimental rats. Furthermore, a highly significant reduction was observed in group G 2 (−17.72%) of experimental rats after 60 days and in group G 1 (−14.14%). Overall, a significant reduction was noted in hyperlipidemic groups of experimental rats. A significant reduction was observed in G h1 (−9.50%), but the reduction was lower than Gh 2 (−17.35%) during the 30-day study trial as compared with the control group.

| Effect on triglyceride
A significant reduction in total triglyceride was noted in G 1 (−6.39%) and G 2 (−11.66%) of normal experimental rats compared to G 0 after 30 days (Table 3). After the 60 days of the experimental trial, a significant reduction was noted in the G 1 and G 2 of normal experimental rats. On the other hand, a reduction in total triglyceride was observed in Gh 1 (−12.67%) and Gh 2 (−13.74%) of hyperlipidemic experimental  Note: Mean ± Standard deviation. Where G 0 : Normal group of experimental rats fed with basal diet. G 1 : Normal group of experimental rats fed with a basal diet supplemented with 125 mg flaxseed polysaccharide gum (FPG). G 2 : Normal group of experimental rats fed with a basal diet supplemented with 250 mg FPG. Gh 0 : Hyperlipidemic group of experimental rats fed with a hypercholesteremic diet. Gh 1 : Hyperlipidemic group of experimental rats fed with a hypercholesteremic diet supplemented with 125 mg FPG. Gh 2 : Hyperlipidemic group of experimental rats fed with a hypercholesteremic diet supplemented with 250 mg FPG. Means showing different uppercase letters represent significant differences (p < .05) within the control or hyperlipidemic experimental groups. Whereas means showing different lowercase letters represent significant differences (p < .05) across a 0-to-60-day trial.
rats compared with baseline data during 30 days of study. The maximum reduction of −25.06% and −22.01% was observed in Gh 2 and Gh 1, respectively, after 60 days of trial. The current research outcomes highlighted the importance of FPG in managing triglyceride levels among all experimental groups (Table 3).
To better understand the effects of FPG on serum triglyceride levels, the studies above concluded that the viscous fiber reduced the postprandial triglyceride level in an animal model (Reimer et al., 2011). Studies have shown no significant association between FPG consumption and its effect on triglycerides. The regulatory mechanism is performed through the triglyceride homeostasis mechanism by lipid droplets (LDs) and smooth endoplasmic reticulum (sER) in the periphery of hepatocytes (Rai et al., 2017). A similar observation has also been noted by Kristensen et al. (2012), who also found a gradually decreasing trend in triglyceride levels in experimental subjects.

| Effect on high-density lipoprotein cholesterol (HDL-c) level
Significant differences were noted in HDL-c levels in experimental groups among all categories of hyperlipidemic rats. A nonsignificant difference was observed in the normal group of experimental rats after 30 days compared with baseline data. The increasing trend was observed in G 2 (10.51%) than in G 1 (5.35%) compared to control group G 0 after 60 days of the study trial. On the other hand, for hyperlipidemic rats maximum increase was observed in Gh 2 (26.19%) than in Gh 1 (20.92%) after 30 days of the study trial. The increasing trend was also observed in Gh 2 (48.96%) and Gh 1 (31.11%), respectively, after 60 days of the study trial (Table 4).
LDL-c, the main indicator of cardiovascular diseases, is converted through oxidation, glycosylation, and carbamylation (Alique et al., 2015). Studies have shown that the increase in LDL-c levels is mainly responsible for atherosclerotic cardiovascular disease (Gao et al., 2017). Higher levels of LDL-c have also been found to accelerate the risk factors of carotid atherosclerosis before the clinical event (Gao et al., 2018).

| Effect on low-density lipoprotein cholesterol (LDL-c) level
There was an increase in the LDL-c level after feeding with a basal diet and a hypercholesteremic diet ( Table 5). On the other hand, after giving a diet supplemented with FPG, a significant reduction in LDL-c levels was observed in experimental rats. Reduction of −2.25% to −4.36% was noted in G 1 and G 2 of normal experimental rats during 30 days, and almost similar results were also noted, even after 60 days of experimental trial (−5.05% to −9.45%) compared with baseline data. On the other hand, the reduction in LDL-c level after 30-60 days of an experimental trial in hyperlipidemic rats was higher in Gh 1 and Gh 2 (−16.44% and-18.82%) after 30 days of the study trial than that of normal groups. After 60 days of the experimental trial, the LDL-c level was reduced to normal, as shown in Table 5.  hyperlipidemia was evaluated in this study. It is quite evident from current research that FPG supplementation improved biomarkers like total cholesterol, triglyceride, LDL-c, and HDL-c. Therefore, based on current research, it was suggested that FPG has antihyperlipidemic potential. Still, the mechanisms of action of FPG as antihyperlipidemic and other therapeutic potential have yet to be explored.

DATA AVA I L A B I L I T Y S TAT E M E N T
The dataset supporting the conclusions of this article is included within the article. Note: Mean ± Standard deviation. Where G 0 : Normal group of experimental rats fed with basal diet. G 1 : Normal group of experimental rats fed with a basal diet supplemented with 125 mg flaxseed polysaccharide gum (FPG). G 2 : Normal group of experimental rats fed with a basal diet supplemented with 250 mg FPG. Gh 0 : Hyperlipidemic group of experimental rats fed with a hypercholesteremic diet. Gh 1 : Hyperlipidemic group of experimental rats fed with a hypercholesteremic diet supplemented with 125 mg FPG. Note: Mean ± Standard deviation. Where G 0 : Normal group of experimental rats fed with basal diet. G 1 : Normal group of experimental rats fed with a basal diet supplemented with 125 mg flaxseed polysaccharide gum (FPG). G 2 : Normal group of experimental rats fed with a basal diet supplemented with 250 mg FPG. Gh 0 : Hyperlipidemic group of experimental rats fed with a hypercholesteremic diet. Gh 1 : Hyperlipidemic group of experimental rats fed with a hypercholesteremic diet supplemented with 125 mg FPG. Gh 2 : Hyperlipidemic group of experimental rats fed with a hypercholesteremic diet supplemented with 250 mg FPG. Means showing different uppercase letters represent significant differences (p < .05) within the control or hyperlipidemic experimental groups. Whereas means showing different lowercase letters represent significant differences (p < .05) across a 0-to-60-day trial.